Genome editing method

ABSTRACT

The present invention relates to the field of genetic engineering. In particular, the present invention relates to a genome editing method with high efficiency and high specificity. More specifically, the present invention relates to a method for increasing the efficiency of site-directed modification of a target sequence in a genome of an organism by a high-specificity Cas9 nuclease variant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International PatentApplication No. PCT/CN2018/076949, filed Feb. 22, 2018, which claimspriority to Chinese Patent Application No. 201710089494.9, filed Feb.20, 2017, both of which applications are herein incorporated byreference in their entireties.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 19, 2019, is named245761_000084seqlist.txt, and is 102,206 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering. Inparticular, the present invention relates to a genome editing methodwith high efficiency and high specificity. More specifically, thepresent invention relates to a method for increasing the efficiency ofsite-directed modification of a target sequence in a genome of anorganism by a high-specificity Cas9 nuclease variant.

BACKGROUND OF THE INVENTION

Clustered regularly interspaced short palindromic repeats and CRISPRassociated system (CRISPR/Cas9) is the most popular tool for genomeediting. In the system, Cas9 protein cleaves a specific DNA sequenceunder the guidance of a gRNA to create a double-strand break (DSB). DSBcan activate intracellular repair mechanisms of non-homologous endjoining (NHEJ) and homologous recombination (HR) to repair DNA damage incells such that the specific DNA sequence is edited during the repairprocess. Currently, the most commonly used Cas9 protein is Cas9 derivedfrom Streptococcus pyogenes (SpCas9). One disadvantage of theCRISPR/Cas9 genome editing system is its low specificity and off-targeteffect, which greatly limit the application thereof.

There remains a need in the art for a method and tool that allow forefficient, high-specific genome editing.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a genome editing systemfor site-directed modification of a target sequence in the genome of acell, which comprises at least one selected from the following i) toiii):

i) a Cas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a tRNA-guide RNA fusion;

ii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a tRNA-guide RNA fusion; and

iii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant and a nucleotide sequence encoding a tRNA-guideRNA fusion;

wherein the Cas9 nuclease variant has higher specificity as comparedwith the wild-type Cas9 nuclease,

wherein the 5′ end of the guide RNA is linked to the 3′ end of the tRNA,

wherein the fusion is cleaved at the 5′ end of the guide RNA after beingtranscribed in the cell, thereby forming a guide RNA that does not carryextra nucleotide at the 5′ end.

In a second aspect, the present invention provides a genome editingsystem for site-directed modification of a target sequence in the genomeof a cell, which comprises at least one selected from the following i)to iii):

i) a Cas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a ribozyme-guide RNA fusion;

ii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a ribozyme-guide RNA fusion; and

iii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant and a nucleotide sequence encoding aribozyme-guide RNA fusion;

wherein the Cas9 nuclease variant has higher specificity as comparedwith the wild-type Cas9 nuclease,

wherein the 5′ end of the guide RNA is linked to the 3′ end of a firstribozyme,

wherein the first ribozyme is designed to cleave the fusion at the 5′end of the guide RNA, thereby forming a guide RNA that does not carryextra nucleotide at the 5′ end.

In a third aspect, the present invention provides a method forgenetically modifying a cell, comprising introducing the genome editingsystem of the present invention into the cell, whereby the Cas9 nucleasevariant is targeted to a target sequence in the genome of the cell bythe guide RNA, and results in substitution, deletion and/or addition ofone or more nucleotides in the target sequence.

In a fourth aspect, the present invention provides a geneticallymodified organism, which comprises a genetically modified cell producedby the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the strategies for designing sgRNA for targetsequences with different 5′ end nucleotides when using U3 or U6. A: byfusion with tRNA, sgRNA can be designed without considering the 5′ endnucleotide of the target sequence; B: precise cleavage of tRNA-sgRNAfusion.

FIG. 2 shows the editing efficiency of WT SpCas9 (wild type SpCas9),eSpCas9(1.0), eSpCas9(1.1), SpCas9-HF1 on targets of class (1).

FIG. 3 shows shows the editing efficiency of WT SpCas9 (wild typeSpCas9), eSpCas9(1.0), eSpCas9(1.1), SpCas9-HF1 on targets of class (2).

FIG. 4 shows that the additional nucleotide at 5′ end of sgRNA affectsthe editing efficiency when U6 promoter is used.

FIGS. 5A and 5B show that for the OsMKK4 locus, tRNA-sgRNA can improvethe editing efficiency and maintain high specificity as compared tosgRNA.

FIGS. 6A and 6B show that for the OsCDKB2 locus, the use of tRNA-sgRNAcan increase the editing efficiency to the level of wild-type SpCas9,while maintaining high specificity.

FIGS. 7A and 7B show the editing specificity of Cas9 variant formismatch between gRNA and target sequence. In FIG. 7A, the sequenceslisted are SEQ ID NOS: 23 and 65-83 in the order shown. In FIG. 7B, thesequences listed are also SEQ ID NOS: 23 and 65-83 in the order shown.

FIG. 8 shows tRNA-sgRNA improved the editing efficiency of eSpCas9(1.1)and SpCas9-HF1 to that of wild-type SpCas9 in human cells.

FIG. 9 shows the sequence structure of pUC57-U3-tRNA-sgRNA vector fortRNA-sgRNA fusion expression.

DETAILED DESCRIPTION OF THE INVENTION 1. Definition

In the present invention, unless indicated otherwise, the scientific andtechnological terminologies used herein refer to meanings commonlyunderstood by a person skilled in the art. Also, the terminologies andexperimental procedures used herein relating to protein and nucleotidechemistry, molecular biology, cell and tissue cultivation, microbiology,immunology, all belong to terminologies and conventional methodsgenerally used in the art. For example, the standard DNA recombinationand molecular cloning technology used herein are well known to a personskilled in the art, and are described in details in the followingreferences: Sambrook, J., Fritsch, E. F. and Maniatis, T., MolecularCloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: ColdSpring Harbor, 1989. In the meantime, in order to better understand thepresent invention, definitions and explanations for the relevantterminologies are provided below.

“Cas9 nuclease” and “Cas9” can be used interchangeably herein, whichrefer to a RNA directed nuclease, including the Cas9 protein orfragments thereof (such as a protein comprising an active DNA cleavagedomain of Cas9 and/or a gRNA binding domain of Cas9). Cas9 is acomponent of the CRISPR/Cas (clustered regularly interspaced shortpalindromic repeats and its associated system) genome editing system,which targets and cleaves a DNA target sequence to form a DNA doublestrand breaks (DSB) under the guidance of a guide RNA.

“guide RNA” and “gRNA” can be used interchangeably herein, whichtypically are composed of crRNA and tracrRNA molecules forming complexesthrough partial complement, wherein crRNA comprises a sequence that issufficiently complementary to a target sequence for hybridization anddirects the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind tothe target sequence. However, it is known in the art that single guideRNA (sgRNA) can be designed, which comprises the characteristics of bothcrRNA and tracrRNA.

As used herein, the terms “tRNA” and “transfer RNA” are usedinterchangeably to refer to small molecule RNAs that have the functionof carrying and transporting amino acids. The tRNA molecule usuallyconsists of a short chain of about 70-90 nucleotides folded into aclover shape. In eukaryotes, tRNA genes in the genome are transcribedinto tRNA precursors, which are then processed into mature tRNA afterexcision of the 5′ and 3′ additional sequences by RNase P and RNase Z.

As used herein, the term “ribozyme” refers to an RNA molecule that has acatalytic function which participates in the cleavage and processing ofRNA by catalyzing the transphosphate and phosphodiester bond hydrolysisreactions.

“Genome” as used herein encompasses not only chromosomal DNA present inthe nucleus, but also organelle DNA present in the subcellularcomponents (e.g., mitochondria, plastids) of the cell.

As used herein, “organism” includes any organism that is suitable forgenomic editing. Exemplary organisms include, but are not limited to,mammals such as human, mouse, rat, monkey, dog, pig, sheep, cattle, cat;poultry such as chicken, duck, goose; plants including monocots anddicots such as rice, corn, wheat, sorghum, barley, soybean, peanut,Arabidopsis and the like.

“Genetically modified organism” or “genetically modified cell” means anorganism or cell that contains an exogenous polynucleotide or modifiedgene or expression control sequence within its genome. For example, theexogenous polynucleotide is stably integrated into the genome of anorganism or cell and inherited for successive generations. The exogenouspolynucleotide can be integrated into the genome alone or as part of arecombinant DNA construct. The modified gene or expression controlsequence is the sequence in the genome of the organism or cell thatcomprises single or multiple deoxynucleotide substitutions, deletionsand additions.

The term “exogenous” with respect to sequence means a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably to refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

As used herein, an “expression construct” refers to a vector suitablefor expression of a nucleotide sequence of interest in an organism, suchas a recombinant vector. “Expression” refers to the production of afunctional product. For example, the expression of a nucleotide sequencemay refer to transcription of the nucleotide sequence (such astranscribe to produce an mRNA or a functional RNA) and/or translation ofRNA into a protein precursor or a mature protein.

“Expression construct” of the invention may be a linear nucleic acidfragment, a circular plasmid, a viral vector, or, in some embodiments,an RNA that can be translated (such as an mRNA).

“Expression construct” of the invention may comprise regulatorysequences and nucleotide sequences of interest that are derived fromdifferent sources, or regulatory sequences and nucleotide sequences ofinterest derived from the same source, but arranged in a mannerdifferent than that normally found in nature.

“Regulatory sequence” or “regulatory element” are used interchangeablyand refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleic acid fragment capable of controlling thetranscription of another nucleic acid fragment. In some embodiments ofthe present invention, the promoter is a promoter capable of controllingthe transcription of a gene in a cell, whether or not it is derived fromthe cell. The promoter may be a constitutive promoter or atissue-specific promoter or a developmentally-regulated promoter or aninducible promoter.

“Constitutive promoter” refers to a promoter that may cause expressionof a gene in most circumstances in most cell types. “Tissue-specificpromoter” and “tissue-preferred promoter” are used interchangeably, andrefer to a promoter that is expressed predominantly but not necessarilyexclusively in one tissue or organ, but that may also be expressed inone specific cell or cell type. “Developmentally regulated promoter”refers to a promoter whose activity is determined by developmentalevents. “Inducible promoter” selectively expresses a DNA sequenceoperably linked to it in response to an endogenous or exogenous stimulus(environment, hormones, or chemical signals, and so on).

As used herein, the term “operably linked” means that a regulatoryelement (for example but not limited to, a promoter sequence, atranscription termination sequence, and so on) is associated to anucleic acid sequence (such as a coding sequence or an open readingframe), such that the transcription of the nucleotide sequence iscontrolled and regulated by the transcriptional regulatory element.Techniques for operably linking a regulatory element region to a nucleicacid molecule are known in the art.

“Introduction” of a nucleic acid molecule (e.g., plasmid, linear nucleicacid fragment, RNA, etc.) or protein into an organism means that thenucleic acid or protein is used to transform a cell of the organism suchthat the nucleic acid or protein functions in the cell. As used in thepresent invention, “transformation” includes both stable and transienttransformations. “Stable transformation” refers to the introduction ofan exogenous nucleotide sequence into the genome, resulting in thestable inheritance of foreign genes. Once stably transformed, theexogenous nucleic acid sequence is stably integrated into the genome ofthe organism and any of its successive generations. “Transienttransformation” refers to the introduction of a nucleic acid molecule orprotein into a cell, performing its function without the stableinheritance of an exogenous gene. In transient transformation, theexogenous nucleic acid sequence is not integrated into the genome.

2. Genome Editing System with High Efficiency and High Specificity

It has been reported that the Cas9 nuclease variant eSpCas9 (1.0)(K810A/K1003A/R1060A), eSpCas9(1.1) (K848A/K1003A/R1060A) of Feng Zhanget al., and the Cas9 nuclease variant SpCas9-HF1(N497A/R661A/Q695A/Q926A) developed by J. Keith Joung et al., arecapable of significantly reducing the off-target rate in genomicediting, and thus have high specificity. However, surprisingly, thepresent inventors found that these three Cas9 nuclease variants, whilehaving high specificity, have a much lower gene editing efficiencycompared to wild-type Cas9.

The present inventors have surprisingly found that by fusing the 5′ endof the guide RNA to a tRNA, the editing efficiency of thehigh-specificity Cas9 nuclease variant can be increased, even to thewild-type level, while maintaining the high specificity.

Not intended to be limited by any theory, it is believed that theediting efficiency reduction of high-specificity Cas9 nuclease variantsis related to whether the transcription of guide RNA can be preciselyinitiated or not. In the art, commonly used promoters for producingguide RNA in vivo include for example U6 or U3 snRNA promoters, forwhich the transcription is driven by RNA polymerase III. U6 promoterneeds to initiate transcription at G, and thus for the target sequenceswith the first nucleotide of A, C or T, an additional G will be presentat 5′ end of sgRNA as transcribed. U3 promoter initiates transcriptionat A, and thus for the target sequences with the first nucleotide of G,C or T, an additional A will be present at 5′ end of sgRNA astranscribed. The inventors found that, the editing efficiency ofhigh-specificity Cas9 nuclease variants is reduced in the case that anadditional nucleotide is present at 5′ end of the sgRNA. By fusiontranscription with a tRNA, due to the mechanism of precisely processingtRNA (precisely removing additional sequence of 5′ and 3′ of tRNAprecursor to form mature tRNA), sgRNA without additional nucleotide at5′ end can be readily obtained even using U6 or U3 promoters, withoutthe need of considering the type of the first nucleotide of the targetsequence. Thereby, the editing efficiency of high specificity Cas9nuclease variants can be improved, and the selectable range of targetsequences can be extended. In addition, not intended to be limited byany theory, fusion with tRNA can increase the expression level of sgRNA,which may also contribute to the improvement of editing efficiency ofhigh-specificity Cas9 nuclease variants.

Therefore, the present invention provides a genome editing system forsite-directed modification of a target sequence in the genome of a cell,which comprises at least one selected from the following i) to iii):

i) a Cas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a tRNA-guide RNA fusion;

ii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a tRNA-guide RNA fusion; and

iii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant and a nucleotide sequence encoding a tRNA-guideRNA fusion;

wherein the Cas9 nuclease variant has higher specificity as comparedwith the wild-type Cas9 nuclease,

wherein the 5′ end of the guide RNA is linked to the 3′ end of the tRNA,

wherein the fusion is cleaved at the 5′ end of the guide RNA after beingtranscribed in the cell, thereby forming a guide RNA that does not carryextra nucleotide at the 5′ end.

In some embodiments, the tRNA and the cell to be modified are from thesame species.

In some specific embodiments, the tRNA is encoded by the followingsequence:aacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgca(SEQ ID NO:1).

The design of the tRNA-guide RNA fusion is within the skill of theperson in the art. For example, reference can be made to Xie et al.,PNAS, Mar. 17, 2015; vol. 112, no. 11, 3570-3575.

The present invention also considers the fusion of a guide RNA and aribozyme. On the basis that it is found in the invention that theediting efficiency of high-specificity Cas9 nuclease variants is relatedto precise transcription initiation of sgRNA, by using the ability ofribozyme to cut RNA at specific site, it is possible to produce sgRNAwithout additional nucleotide at 5′ end by rational design of a fusionof RNA and ribozyme, so as to improve editing efficiency while maintainthe high specificity.

Therefore, the invention also provides a genome editing system forsite-directed modification of a target sequence in the genome of a cell,which comprises at least one selected from the following i) to iii):

i) a Cas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a ribozyme-guide RNA fusion;

ii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a ribozyme-guide RNA fusion; and

iii) an expression construct comprising a nucleotide sequence encoding aCas9 nuclease variant and a nucleotide sequence encoding aribozyme-guide RNA fusion;

wherein the Cas9 nuclease variant has higher specificity as comparedwith the wild-type Cas9 nuclease,

wherein the 5′ end of the guide RNA is linked to the 3′ end of a firstribozyme,

wherein the first ribozyme is designed to cleave the fusion at the 5′end of the guide RNA, thereby forming a guide RNA that does not carryextra nucleotide at the 5′ end.

In one embodiment, the 3′ end of the guide RNA is linked to the 5′ endof a second ribozyme, the second ribozyme is designed to cleave thefusion at the 3′ end of the guide RNA, thereby forming a guide RNA thatdoes not carry extra nucleotide at the 3′ end.

The design of the first ribozyme or the second ribozyme is within theskill of the person in the art. For example, reference can be made toGao et al., JIPB, April, 2014; Vol 56, Issue 4, 343-349.

In one specific embodiment, the first ribozyme is encoded by thefollowing sequence: 5′-(N)₆CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC-3′ (SEQID NO:12), wherein N is independently selected from A, G, C, and T, and(N)₆ refers to a sequence reversely complementary to the first 6nucleotides at 5′ end of the guide RNA. In one specific embodiment, thesecond ribozyme is encoded by the following sequence: 5′-GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGAC-3′ (SEQ ID NO:13).

The Cas9 nuclease variant in the invention that has higher specificityas compared with wild type Cas9 nuclease can be derived from Cas9 ofvarious species, for example, derived from Cas9 of Streptococcuspyogenes (SpCas9, nucleotide sequence shown in SEQ ID NO:2, amino acidsequence shown in SEQ ID NO:3).

In some embodiments of the invention, the Cas9 nuclease variant is avariant of SEQ ID NO:2, which comprises an amino acid substitution atposition 855 of SEQ ID NO:2. In some specific embodiments, the aminoacid substitution at position 855 is K855A.

In some embodiments of the invention, the Cas9 nuclease variant is avariant of SEQ ID NO:2, which comprises amino acid substitutions atpositions 810, 1003 and 1060 of SEQ ID NO:2. In some specificembodiments, the amino acid substitutions respectively are K810A, K1003Aand R1060A.

In some embodiments of the invention, the Cas9 nuclease variant is avariant of SEQ ID NO:2, which comprises amino acid substitutions atpositions 848, 1003 and 1060 of SEQ ID NO:2. In some specificembodiments, the amino acid substitutions respectively are K848A, K1003Aand R1060A.

In some embodiments of the invention, the Cas9 nuclease variant is avariant of SEQ ID NO:2, which comprises amino acid substitutions atpositions 611, 695 and 926 of SEQ ID NO:2. In some specific embodiments,the amino acid substitutions respectively are R611A, Q695A and Q926A.

In some embodiments of the invention, the Cas9 nuclease variant is avariant of SEQ ID NO:2, which comprises amino acid substitutions atpositions 497, 611, 695 and 926 of SEQ ID NO:2. In some specificembodiments, the amino acid substitutions respectively are N497A, R611A,Q695A and Q926A.

In some specific embodiments of the invention, the Cas9 nuclease variantcomprises an amino acid sequence as shown in SEQ ID NO:4 (eSpCas9(1.0)),SEQ ID NO:5 (eSpCas9(1.1)) or SEQ ID NO:6 (SpCas9-HF1).

In some embodiments of the invention, the Cas9 nuclease variant of theinvention further comprises a nuclear localization sequence (NLS). Ingeneral, one or more NLSs in the Cas9 nuclease variant should havesufficient strength to drive the accumulation of the Cas9 nucleasevariant in the nucleus of the cell in an amount sufficient for thegenome editing function. In general, the strength of the nuclearlocalization activity is determined by the number and position of NLSs,and one or more specific NLSs used in the Cas9 nuclease variant, or acombination thereof.

In some embodiments of the present invention, the NLSs of the Cas9nuclease variant of the invention may be located at the N-terminusand/or the C-terminus. In some embodiments, the Cas9 nuclease variantcomprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In someembodiments, the Cas9 nuclease variant comprises about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the N-terminus. In someembodiments, the Cas9 nuclease variant comprises about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the C-terminus. In someembodiments, the Cas9 nuclease variant comprises a combination of these,such as one or more NLSs at the N-terminus and one or more NLSs at theC-terminus. Where there are more than one NLS, each NLS may be selectedas independent from other NLSs. In some preferred embodiments of theinvention, the Cas9 nuclease variant comprises two NLSs, for example,the two NLSs are located at the N-terminus and the C-terminus,respectively.

In general, NLS consists of one or more short sequences of positivelycharged lysine or arginine exposed on the surface of a protein, butother types of NLS are also known in the art. Non-limiting examples ofNLSs include KKRKV (nucleotide sequence 5′-AAGAAGAGAAAGGTC-3′ (SEQ IDNO: 14)), PKKKRKV (nucleotide sequence 5′-CCCAAGAAGAAGAGGAAGGTG-3′ (SEQID NO: 15) or CCAAAGAAGAAGAGGAAGGTT (SEQ ID NO: 16), or SGGSPKKKRKV (SEQID NO: 17) (nucleotide sequence 5′-TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG-3′)(SEQ ID NO: 18.

In some embodiments of the invention, the N-terminus of the Cas9nuclease variant comprises an NLS with an amino acid sequence shown byPKKKRKV (SEQ ID NO: 19). In some embodiments of the invention, theC-terminus of the Cas9 nuclease variant comprises an NLS with an aminoacid sequence shown by SGGSPKKKRKV (SEQ ID NO: 17).

In addition, the Cas9 nuclease variant of the present invention may alsoinclude other localization sequences, such as cytoplasmic localizationsequences, chloroplast localization sequences, mitochondriallocalization sequences, and the like, depending on the location of theDNA to be edited.

For obtaining effective expression in the target cell, in someembodiments of the invention, the nucleotide sequence encoding the Cas9nuclease variant is codon-optimized for the organism where the cell tobe genome-edited is from.

Codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g. about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/ and these tables canbe adapted in a number of ways. See Nakamura, Y., et al.“Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000).

The organism, from which the cell that can be genome edited by thesystem of the invention is derived, includes but is not limited to,mammals such as human, mice, rat, monkey, dog, pig, sheep, cow and cat;poultry such as chicken, duck and goose; plants including monocotyledonsand dicotyledons, e.g. rice, maize, wheat, sorghum, barley, soybean,peanut and Arabidopsis thaliana and the like.

In some specific embodiments of the invention, the codon-optimizednucleotide sequence encoding the Cas9 nuclease variant is as shown inSEQ ID NO:7 (eSpCas9(1.0)), SEQ ID NO:8 (eSpCas9(1.1)) or SEQ ID NO:9(SpCas9-HF1).

In some embodiments of the invention, the guide RNA is a single guideRNA (sgRNA). Methods of constructing suitable sgRNAs according to agiven target sequence are known in the art. See e.g., Wang, Y. et al.Simultaneous editing of three homoeoalleles in hexaploid bread wheatconfers heritable resistance to powdery mildew. Nat. Biotechnol. 32,947-951 (2014); Shan, Q. et al. Targeted genome modification of cropplants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013);Liang, Z. et al. Targeted mutagenesis in Zea mays using TALENs and theCRISPR/Cas system. J Genet Genomics. 41, 63-68 (2014).

In some embodiments of the invention, the nucleotide sequence encodingthe Cas9 nuclease variant and/or the nucleotide sequence encoding theguide RNA fusion are operatively linked to an expression regulatoryelement such as a promoter.

Examples of promoters that can be used in the present invention includebut are not limited to polymerase (pol) I, pol II or pol III promoters.Examples of pol I promoters include chicken RNA pol I promoter. Examplesof pol II promoters include but are not limited to cytomegalovirusimmediate early(CMV) promoter, rous sarcoma virus long terminal repeat(RSV-LTR) promoter and simian virus 40 (SV40) immediate early promoter.Examples of pol III promoters include U6 and H1 promoter. Induciblepromoter such as metalothionein promoter can be used. Other examples ofpromoters include T7 bacteriophage promoter, T3 bacteriophage promoter,β-galactosidase promoter and Sp6 bacteriophage promoter etc. When usedfor plants, promoters that can be used include but are not limited tocauliflower mosaic virus 35S promoter, maize Ubi-1 promoter, wheat U6promoter, rice U3 promoter, maize U3 promoter and rice actin promoteretc.

3. Method for Genetically Modifying a Cell

In another aspect, the invention provides a method for geneticallymodifying a cell, comprising: introducing the genome editing system ofthe invention to the cell, thereby the Cas9 nuclease variant is targetedto the target sequence in the genome of the cell by the guide RNA, andresults in substitution, deletion and/or addition of one or morenucleotides in the target sequence.

The design of the target sequence that can be recognized and targeted bya Cas9 and guide RNA complex is within the technical skills of one ofordinary skill in the art. In general, the target sequence is a sequencethat is complementary to a leader sequence of about 20 nucleotidescomprised in guide RNA, and the 3′-end of which is immediately adjacentto the protospacer adjacent motif (PAM) NGG.

For example, in some embodiments of the invention, the target sequencehas the structure: 5′-Nx-NGG-3′, wherein N is selected independentlyfrom A, G, C, and T; X is an integer of 14≤X≤30; NX represents Xcontiguous nucleotides, and NGG is a PAM sequence. In some specificembodiments of the invention, X is 20.

In the present invention, the target sequence to be modified may belocated anywhere in the genome, for example, within a functional genesuch as a protein-coding gene or, for example, may be located in a geneexpression regulatory region such as a promoter region or an enhancerregion, and thereby accomplish the functional modification of said geneor accomplish the modification of a gene expression.

The substitution, deletion and/or addition in the target sequence of thecell can be detected by T7EI, PCR/RE or sequencing methods, see e.g.,Shan, Q., Wang, Y., Li, J. & Gao, C. Genome editing in rice and wheatusing the CRISPR/Cas system. Nat. Protoc. 9, 2395-2410 (2014).

In the method of the present invention, the genome editing system can beintroduced into the cell by using various methods well known by theskilled in the art.

Methods for introducing the genome editing system of the presentinvention into the cell include, but are not limited to calciumphosphate transfection, protoplast fusion, electroporation, liposometransfection, microinjection, viral infection (such as a baculovirus, avaccinia virus, an adenovirus and other viruses), particle bombardment,PEG-mediated protoplast transformation or agrobacterium-mediatedtransformation.

The cell which can be subjected to genome editing with the method of thepresent invention can be from, for example, mammals such as human,mouse, rat, monkey, dog, pig, sheep, cow and cat; poultry such aschicken, duck and goose; and plants including monocotyledons anddicotyledons such as rice, maize, wheat, sorghum, barley, soybean,peanut and Arabidopsis thaliana etc.

In some embodiments, the method of the present invention is performed invitro. For example, the cell is an isolated cell. In some otherembodiments, the method of the present invention can be performed invivo. For example, the cell is a cell within an organism, and the systemof the present invention can be introduced in-vivo into said cell byusing, for example, a virus-mediated method. In some embodiments, thecell is a germ cell. In some implementations, the cell is a somaticcell.

In another aspect, the present invention further provides a geneticallymodified organism comprising a genetically modified cell produced by themethod of the present invention.

The organism includes, but is not limited to mammals such as humans,mice, rats, monkeys, dogs, pigs, sheep, cows and cats; poultry such aschicken, ducks and geese; and plants including monocotyledons anddicotyledons such as rice, maize, wheat, sorghum, barley, soybean,peanuts and Arabidopsis thaliana.

EXAMPLES Materials and Methods Construction of Binary Expression VectorspJIT163-SpCas9, PJIT163-eSpCas9(1.0), pJIT163-eSpCas9(1.1) andpJIT163-SpCas9-HF1

SpCas9, eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 sequences werecodon-optimized for rice. SpCas9, eSpCas9(1.0), eSpCas9(1.1) andSpCas9-HF1 were obtained by site-directed mutagenesis using FastMultiSite Mutagenesis System (TransGen) with pJIT163-SpCas9 plasmid (SEQID NO:10) as the template.

Construction of sgRNA Expression Vector

sgRNA target sequences used in the experiments are showed in table 1 asfollows:

TABLE 1 Target Gene and sgRNA Target Sequence sgRNA Target sequenceOligo-F Oligo-R OsCDKB2 AGGTCGGGGAGGGGA GGCAAGGTCGGGGAGGAAACGTACGTCCCCTCC CGTACGGG (SEQ ID NO: GGACGTAC (SEQ ID NO:CCGACCT (SEQ ID NO: 20) 21) 22) OsMKK4 GACGTCGGCGAGGAA GGCAGACGTCGGCGAGAAACAGGCCTTCCTCGC GGCCTCGG (SEQ ID NO: GAAGGCCT (SEQ IN NO:CGACGTC (SEQ ID NO: 23) 24) 25) A1 CATGGTGGGGAAAGCT GGCACATGGTGGGGAAAAAACTCCAAGCTTTCCC TGGAGGG (SEQ ID NO: GCTTGGA (SEQ ID NO:CACCATG (SEQ ID NO: 26) 27) 28) A2 CCGGACGACGACGTCG GGCACCGGACGACGACAAACTCGTCGACGTCGT ACGACGG (SEQ ID NO: GTCGACGA (SEQ ID NO:CGTCCGG (SEQ ID NO: 29) 30) 31) A3 TTGAAGTCCCTTCTAGA GGCATTGAAGTCCCTTCTAAACCCATCTAGAAGGG TGGAGG (SEQ ID NO: AGATGG (SEQ ID NO:ACTTCAA (SEQ ID NO: 32) 33) 34) A4 ACTGCGACACCCAGAT GGCAACTGCGACACCCAAAACCGATATCTGGGTG ATCGTGG (SEQ ID NO: GATATCG (SEQ ID NO:TCGCAGT (SEQ ID NO: 35) 36) 37) PDS GTTGGTCTTTGCTCCTG GGCAGTTGGTCTTTGCTAAACCTGCAGGAGCAA CAGAGG (SEQ ID NO: CCTGCAG (SEQ ID NO:AGACCAAC (SEQ ID NO: 38) 39) 40)

sgRNA expression vectors: pOsU3-CDKB2-sgRNA, pOsU3-MKK4-sgRNA,pOsU3-A1-sgRNA as well as pOsU3-A2-sgRNA, pOsU3-A3-sgRNA, pOsU3-A4-sgRNAand pOsU3-PDS-sgRNA are constructed on the basis of pOsU3-sgRNA(AddgeneID53063) as described previously (Shan, Q. et al. Targeted genomemodification of crop plants using a CRISPR-Cas system. Nat. Biotechnol.31, 686-688, 2013).

Construction of tRNA-sgRNA Expression Vectors

tRNA-sgRNA expression vectors are constructed on the basis of thepUC57-U3-tRNA-sgRNA vector (SEQ ID NO:11, FIG. 6). A linear vector isobtained after digestion of pUC57-U3-tRNA-sgRNA with BsaI, thecorresponding oligo-F and oligo-R are annealed and connected into thelinear vector, and the subsequent steps are similar to the constructionof the sgRNA expression vectors.

TABLE 2Target Genes and Oligonucleotide Sequences for Constructing tRNA-sgRNAExpression Vectors sgRNA Target sequence Oligo-F Oligo-R OsCDKB2AGGTCGGGGAGGGGA TGCAAGGTCGGGGAGG AAACGTACGTCCCCTCC CGTACGGG (SEQ IDGGACGTAC (SEQ ID NO: CCGACCT (SEQ ID NO: NO: 20) 41) 22) OsMKK4GACGTCGGCGAGGAA TGCAGACGTCGGCGAGG AAACAGGCCTTCCTCGC GGCCTCGG (SEQ IDAAGGCCT (SEQ ID NO: CGACGTC (SEQ ID NO: NO: 23) 42) 25) A1CATGGTGGGGAAAGCT TGCACATGGTGGGGAAA AAACTCCAAGCTTTCCC TGGAGGG (SEQ ID NO:GCTTGGA (SEQ ID NO: CACCATG (SEQ ID NO: 26) 43) 28) A2 CCGGACGACGACGTCGTGCACCGGACGACGACG AAACTCGTCGACGTCGT ACGACGG (SEQ ID NO:TCGACGA (SEQ ID NO: CGTCCGG (SEQ ID NO: 29) 44) 31) A3 TTGAAGTCCCTTCTAGTGCATTGAAGTCCCTTCT AAACCCATCTAGAAGGG ATGGAGG (SEQ ID NO:AGATGG (SEQ ID NO: 45) ACTTCAA (SEQ ID NO: 32) 34) A4 ACTGCGACACCCAGATTGCACTGCGACACCCAG AAACCGATATCTGGGTG ATCGTGG (SEQ ID NO:ATATCG (SEQ ID NO: 46) TCGCAGT (SEQ ID NO: 35) 37) PDS GTTGGTCTTTGCTCCTTGCAGTTGGTCTTTGCTC AAACCTGCAGGAGCAA GCAGAGG (SEQ ID NO:CTGCAG (SEQ ID NO: 47) AGACCAAC (SEQ ID NO: 38) 40)

Protoplast Assays

Rice cultivar nipponbare is used in the research. Protoplaststransformation is performed as described below. Transformation iscarried out with 10 μg of each plasmid by PEG-mediated transfection.Protoplasts were collected after 48 h and DNA was extracted for PCR-REassay.

Preparation and Transformation of Rice Protoplast

1) Leaf sheath of the seedlings were used for protoplasts isolation, andcut into about 0.5 mm wide with a sharp blade.

2) Immediately after incision, transfered into 0.6M Mannitol solution,and placed in the dark for 10 min.

3) Mannitol solution was removed by filtration, and the products weretransfered into enzymolysis solution, and evacuated for 30 min.

4) Enzymolysis was performed for 5-6h in darkness with gently shaking(decolorization shaker, speed 10).

5) After enzymolysis completion, an equal volume of W5 was added,horizontal shake for 10s to release protoplasts.

6) Protoplasts were filtered into a 50 ml round bottom centrifuge tubewith a 40 μm nylon membrane and washed with W5 solution.

7) 250 g horizontal centrifugation for 3 min to precipitate theprotoplasts, the supernatant was discarded.

8) Protoplasts were resuspended by adding 10 ml W5, and then centrifugedat 250 g for 3 min, and the supernatant was discarded.

9) An appropriate amount of MMG solution was added to resuspend theprotoplasts to a concentration of 2×10⁶/ml.

Note: All the above steps were carried out at room temperature.

10) 10-20 μg plasmid, 200 μl protoplasts (about 4×10⁵ cells), and 220 μlfresh PEG solution were added into a 2 ml centrifugal tube, mixed, andplaced at room temperature in darkness for 10-20 minutes to inducetransformation.

11) After the completion of the transformation, 880 μl W5 solution wasslowly added, and the tubes were gently turned upside down for mixing,250 g horizontal centrifuged for 3 min, and the supernatant wasdiscarded.

12) The products were resuspended in 2 ml WI solution, transfered to asix-well plate, cultivated in room temperature (or 25° C.) in darkness.For protoplast genomic DNA extraction, the products need to becultivated for 48 h.

Mutation Identification by Deep Sequencing

Deep sequencing analysis is performed by reference to Liang, Z., Chen,K., Li, T., Zhang, Y., Wang, Y., Zhao, Q., Liu, J., Zhang, H., Liu, C.,Ran, Y., et al. (2017). Efficient DNA-free genome editing of bread wheatusing CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications 8,14261.

Example 1: Comparing Editing Capacities of WT SpCas9 and VariantsThereof to Target Sites

WT SpCas9, eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 were respectivelyconstructed in a transient expression vector pJIT163, and theexpressions of WT SpCas9, eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 aredriven by a maize ubiquitin gene promoter. sgRNAs were constructed inthe pOsU3-sgRNA vector, and the expression of sgRNAs is driven by OsU3promoter. Rice protoplasts were transformed, and protoplast DNA wasextracted for PCR-RE analysis to evaluate the mutation efficiency. Fivetarget sites (A1, A2, A3, A4 and PDS, see FIG. 2 and FIG. 3) areselected to compare the difference of editing capacities of wild-typeSpCas9 and eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1.

The OsU3 promoter has to initiate transcription with the nucleotide A,and therefore, the design of the sgRNA expression vectors for the targetsites can be divided into two conditions as follows:

(1) If the first nucleotide at the 5′ end of the desired sgRNA targetsequence (20 bp) is any one of G/T/C, as the U3 promoter initiatestranscription with an A, an additional A will be added to the 5′ end ofthe transcribed sgRNA, and furthermore, the transcribed sgRNA cannotcompletely match with the target sequence. sgRNA expression vector canbe constructed as U3+AN₂₀ in FIG. 1, while N₂₀ is the target sequence, Ais the additional nucleotide at 5′ end.

(2) If the first nucleotide at the 5′ end of the desired sgRNA targetsequence (20 bp) is A, it can used by the U3 promoter for initiatingtranscription, and therefore no additional nucleotide will exist at the5′ end of the transcribed sgRNA. sgRNA expression vector can beconstructed as U3+AN₁₉ in FIG. 1, while AN₁₉ is the target sequence.

The selected target sites A1, A2, A3 and PDS belong to target sites ofclass (1), and target site A4 belongs to target sites of class (2).

The experiment results show (FIG. 2) that the editing efficiencies ofeSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 for the target sites of class(1) are extremely low. The difference of the editing efficiencies ofeSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 and the editing efficiency ofWT SpCas9 is not significant for target sites of class (2). This showsthat the additional nucleotide at the 5′ end of the sgRNA resulted fromthe transcription can reduce the editing efficiencies of eSpCas9(1.0),eSpCas9(1.1) and SpCas9-HF1.

Similar to OsU3 promoter, maize U6 promoter (TaU6) has to initiatetranscription with the nucleotide G, and therefore, the design of thesgRNA expression vectors for the target sites can be divided into twoconditions as follows:

(1) If the first nucleotide at the 5′ end of the desired sgRNA targetsequence (20 bp) is any one of A/T/C, as the U6 promoter initiatestranscription with a G, an additional G will be added to the 5′ end ofthe transcribed sgRNA, and furthermore, the transcribed sgRNA cannotcompletely match with the target sequence.

(2) If the first nucleotide at the 5′ end of the desired sgRNA targetsequence (20 bp) is it can used by the U6 promoter for initiatingtranscription, and therefore no additional nucleotide will exist at the5′ end of the transcribed sgRNA.

The OsPDS target site belongs to target sites of class (2). TaU6promoter was used to drive the transcription of GN₁₉ and GN₂₀ sgRNAsagainst OsPDS target site, where GN₂₀ can mimic the target sites ofclass (1), namely with an additional G at 5′ end of the sgRNA.

TABLE 3Target gene and oligonucleotide sequences for construction of TaU6-sgRNAexpression vectors sgRNA Target sequence Oligo-F Oligo-R OsPDS-GN₁₉GTTGGTCTTTGCTCC GGCGTTGGTCTTTGCTC AAACCTGCAGGAGCAA TGCAGAGG (SEQ IDCTGCAG (SEQ ID NO: AGACCAA (SEQ ID NO: NO. 38) 48) 40) OsPDS-GN₂₀GTTGGTCTTTGCTCC GGCGGTTGGTCTTTGCT AAACCTGCAGGAGCAA TGCAGAGG (SEQ IDCCTGCAG (SEQ ID NO: AGACCAAC (SEQ ID NO. 38) 49) NO: 40)

The results show (FIG. 2) that one additional G at 5′ end of the sgRNAsignificantly reduces the editing efficiency of eSpCas9(1.0),eSpCas9(1.1) and SpCas9-HF1.

Example 2: Increasing Editing Efficiency of Cas9 Variants by tRNA-sgRNAFusion

According to the result of the Example 1, an important factorinfluencing the editing efficiencies of eSpCas9(1.0), eSpCas9(1.1) andSpCas9-HF1is weather the sgRNA is precisely initiated or not. Accordingto previous report, fusion of a tRNA to the 5′ end of an sgRNA mayup-regulate the expression of the sgRNA and result in precise cleavageat the 5′ end of the sgRNA, and thereby avoiding additional nucleotideat the 5′ end of the sgRNA. (See Xie K, Minkenberg B, Yang Y. BoostingCRISPR/Cas9 multiplex editing capability with the endogenoustRNA-processing system. Proc Natl Acad Sci USA. 2015 Mar. 17;112(11):3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar. 2.)

sgRNA for each target site in Example 1 was fused to tRNA and expressedunder the control of the OsU3 promoter. Experiments were performed bythe method in Example 1 with tRNA-sgRNAs instead of sgRNAs. As shown inFIG. 2, for the target sites A1, A2, A3 and PDS, the editingefficiencies of eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 aresignificantly increased using tRNA-sgRNAs instead of sgRNAs.

Example 3: Influences of tRNA-sgRNA Fusion to Editing Specificity ofCas9 Variants 3.1 Rice OsMKK4 Target Site

A target site GACGTCGGCGAGGAAGGCCTCGG (SEQ ID NO: 23) in rice gene MKK4was selected to design sgRNA and tRNA-sgRNA. This target site has twopossible off-target sites as shown in FIG. 5. A vector for expressingsgRNA or tRNA-sgRNA and vectors for expressing WTSpCas9, eSpCas9(1.0),eSpCas9(1.1) and SpCas9-HF1 were respectively co-transformed into riceprotoplasts. Two days after transformation, protoplast DNA wasextracted, and genomic fragments of the target site and the off-targetsites were amplified by using specific primers. Mutation rates of thethree sites were analyzed by using second-generation sequencingtechnology.

The experiment result is shown in FIG. 5:

When sgRNAs were used, compared with WTSpCas9, eSpCas9(1.0),eSpCas9(1.1) and SpCas9-HF1 have extremely low off-target effect, buthave significantly lower editing efficiencies.

When tRNA-sgRNAs were used, the editing efficiency of each group wasincreased, however, eSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 canmaintain relatively high specificity. Particularly for SpCas9-HF1, onlyextremely low-level mutation can be detected for both two off-targetsites. Therefore, the combination of tRNA-sgRNA and SpCas9-HF1 isparticularly suitable for genome editing with high efficiency and highspecificity.

3.2 Rice OsCDKB2 Target Site

A target site AGGTCGGGGAGGGGACGTACGGG (SEQ ID NO: 20) in rice geneOsCDKB2 was selected to design sgRNA. This target site has threepossible off-target sites as shown in FIG. 6. A vector for expressingsgRNA or tRNA-sgRNA and vectors for expressing WTSpCas9, eSpCas9(1.0),eSpCas9(1.1) and SpCas9-HF1 were respectively co-transformed into riceprotoplasts. Two days after transformation, protoplast DNA wasextracted, and genomic fragments of the target site and the off-targetsites were amplified by using specific primers. Mutation rates of thefour sites were analyzed by deep sequencing.

The experiment results are shown in FIG. 6. The editing efficiencies ofeSpCas9(1.0), eSpCas9(1.1) and SpCas9-HF1 to the target sites areeffectively increased by using tRNA-sgRNA instead of sgRNA. Inparticular, the editing efficiency of SpCas9-HF1 can be restored to awild-type level, and high specificity can be maintained. As this targetsequence starts with an A, by which the U3 promoter can preciselyinitiate transcription, the increased editing efficiency may result fromthe increased expression level of sgRNA due to the fusion with tRNA.

Example 4: Editing Specificity of Cas9 Variants to Mismatch Between gRNAand Target Sequence

When designing sgRNA for a target site GACGTCGGCGAGGAAGGCCTCGG (SEQ IDNO: 23) in rice gene MKK4, mismatches of two adjacent bases wereartificially introduced (purine for purine, and pyrimidine forpyrimidine). Edition under the condition that sgRNA cannot completelymatch with the target site was detected. It is considered as off-targetif edition can be detected. The experiments were performed in a waysimilar to that in Example 3.1.

The experiment results were shown in FIG. 7. When tRNA-sgRNA is used,SpCas9 variants showed higher sensitivity to mismatches between gRNA andthe target sequence (particularly the mismatch closer to either ends).

Example 5: Editing Efficiency and Specificity of Cas9 Variants in HumanEmbryonic Kidney 293 Cells

sgRNAs were designed against a target sequence GGTGAGTGAGTGTGTGCGTGTGG(SEQ ID NO: 50) within human VEGFA gene. U6:sgRNA-GN₁₉ andU6:tRNA-sgRNA-N₂₀ represent that the sgRNAs transcribed with U6 promoterare 20 nt in length and completely match the target sequence;U6:sgRNA-GN₂₀ represents that the sgRNA transcribed with U6 promoter is21 nt in length and contains an additional G at 5′ end.

The T7E1 assay results show (FIG. 8) that WT Cas9 exhibits similarediting efficiency when sgRNA transcribed with different strategies wereused. However, the editing efficiency of eSpCas9(1.1) and SpCas9-HF1were significantly reduced when the sgRNA contains an additionalnucleotide at 5′end. And by using tRNA-sgRNA fusions, the editingefficiency of eSpCas9(1.1) and SpCas9-HF1 were increased to that of WTCas9 or even higher.

With respect to editing specificity, WT Cas9 resulted in off-targetediting in both sites off target1 and off target2. eSpCas9(1.1) andSpCas9-HF1 did not result in off-target editing when tRNA-sgRNA fusionswere used.

TABLE 4Target gene and oligonucleotide sequences for construction of sgRNAexpression vectors sgRNA Target sequence Oligo-F Oligo-R VEGFA-GN₁₉GGTGAGTGAGTGTGT CACCGGTGAGTGAGTG AAACCACGCACACACTC GCGTGTGG (SEQ IDTGTGCGTG (SEQ ID ACTCACC (SEQ ID NO: NO: 50) NO: 51) 52) VEGFA-GN₂₀GGTGAGTGAGTGTGT CACCGGGTGAGTGAGT AAACCACGCACACACTC GCGTGTGG (SEQ IDGTGTGCGTG (SEQ ID ACTCACCC (SEQ ID NO: NO: 50) NO: 53) 54) VEGFA-tRNA-GGTGAGTGAGTGTGT CACCGaacaaagcaccagtggt AAACCACGCACACACTC N₂₀GCGTGTGG (SEQ ID ctagtggtagaatagtaccctgccac ACTCACCtgcaccagccgggaatNO: 50) ggtacagacccgggttcgattcccg cgaacccgggtctgtaccgtggcaggggctggtgcaGGTGAGTGAG tactattctaccactagaccactggtgctt TGTGTGCGTG (SEQtgttC (SEQ ID NO: 56) ID NO: 55)

Sequence listing tRNA encoding sequence SEQ ID NO: 1aacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgcaSpCas9 nucleotide sequence SEQ ID NO: 2atggcccctaagaagaagagaaaggtcggtattcacggcgttcctgcggcgatggacaagaagtatagtattggtctggacattgggacgaattccgttggctgggccgtgatcaccgatgagtacaaggtcccttccaagaagtttaaggttctggggaacaccgatcggcacagcatcaagaagaatctcattggagccctcctgttcgactcaggcgagaccgccgaagcaacaaggctcaagagaaccgcaaggagacggtatacaagaaggaagaataggatctgctacctgcaggagattttcagcaacgaaatggcgaaggtggacgattcgttctttcatagattggaggagagtttcctcgtcgaggaagataagaagcacgagaggcatcctatctttggcaacattgtcgacgaggttgcctatcacgaaaagtaccccacaatctatcatctgcggaagaagcttgtggactcgactgataaggcggaccttagattgatctacctcgctctggcacacatgattaagttcaggggccattttctgatcgagggggatcttaacccggacaatagcgatgtggacaagttgttcatccagctcgtccaaacctacaatcagctctttgaggaaaacccaattaatgcttcaggcgtcgacgccaaggcgatcctgtctgcacgcctttcaaagtctcgccggcttgagaacttgatcgctcaactcccgggcgaaaagaagaacggcttgttcgggaatctcattgcactttcgttggggctcacaccaaacttcaagagtaattttgatctcgctgaggacgcaaagctgcagctttccaaggacacttatgacgatgacctggataaccttttggcccaaatcggcgatcagtacgcggacttgttcctcgccgcgaagaatttgtcggacgcgatcctcctgagtgatattctccgcgtgaacaccgagattacaaaggccccgctctcggcgagtatgatcaagcgctatgacgagcaccatcaggatctgacccttttgaaggctttggtccggcagcaactcccagagaagtacaaggaaatcttctttgatcaatccaagaacggctacgctggttatattgacggcggggcatcgcaggaggaattctacaagtttatcaagccaattctggagaagatggatggcacagaggaactcctggtgaagctcaatagggaggaccttttgcggaagcaaagaactttcgataacggcagcatccctcaccagattcatctcggggagctgcacgccatcctgagaaggcaggaagacttctacccctttcttaaggataaccgggagaagatcgaaaagattctgacgttcagaattccgtactatgtcggaccactcgcccggggtaattccagatttgcgtggatgaccagaaagagcgaggaaaccatcacaccttggaacttcgaggaagtggtcgataagggcgcttccgcacagagcttcattgagcgcatgacaaattttgacaagaacctgcctaatgagaaggtccttcccaagcattccctcctgtacgagtatttcactgtttataacgaactcacgaaggtgaagtatgtgaccgagggaatgcgcaagcccgccttcctgagcggcgagcaaaagaaggcgatcgtggaccttttgtttaagaccaatcggaaggtcacagttaagcagctcaaggaggactacttcaagaagattgaatgcttcgattccgttgagatcagcggcgtggaagacaggtttaacgcgtcactggggacttaccacgatctcctgaagatcattaaggataaggacttcttggacaacgaggaaaatgaggatatcctcgaagacattgtcctgactcttacgttgtttgaggatagggaaatgatcgaggaacgcttgaagacgtatgcccatctcttcgatgacaaggttatgaagcagctcaagagaagaagatacaccggatggggaaggctgtcccgcaagcttatcaatggcattagagacaagcaatcagggaagacaatccttgactttttgaagtctgatggcttcgcgaacaggaattttatgcagctgattcacgatgactcacttactttcaaggaggatatccagaaggctcaagtgtcgggacaaggtgacagtctgcacgagcatatcgccaaccttgcgggatctcctgcaatcaagaagggtattctgcagacagtcaaggttgtggatgagcttgtgaaggtcatgggacggcataagcccgagaacatcgttattgagatggccagagaaaatcagaccacacaaaagggtcagaagaactcgagggagcgcatgaagcgcatcgaggaaggcattaaggagctggggagtcagatccttaaggagcacccggtggaaaacacgcagttgcaaaatgagaagctctatctgtactatctgcaaaatggcagggatatgtatgtggaccaggagttggatattaaccgcctctcggattacgacgtcgatcatatcgttcctcagtccttccttaaggatgacagcattgacaataaggttctcaccaggtccgacaagaaccgcgggaagtccgataatgtgcccagcgaggaagtcgttaagaagatgaagaactactggaggcaacttttgaatgccaagttgatcacacagaggaagtttgataacctcactaaggccgagcgcggaggtctcagcgaactggacaaggcgggcttcattaagcggcaactggttgagactagacagatcacgaagcacgtggcgcagattctcgattcacgcatgaacacgaagtacgatgagaatgacaagctgatccgggaagtgaaggtcatcaccttgaagtcaaagctcgtttctgacttcaggaaggatttccaattttataaggtgcgcgagatcaacaattatcaccatgctcatgacgcatacctcaacgctgtggtcggaacagcattgattaagaagtacccgaagctcgagtccgaattcgtgtacggtgactataaggtttacgatgtgcgcaagatgatcgccaagtcagagcaggaaattggcaaggccactgcgaagtatttcttttactctaacattatgaatttctttaagactgagatcacgctggctaatggcgaaatccggaagagaccacttattgagaccaacggcgagacaggggaaatcgtgtgggacaaggggagggatttcgccacagtccgcaaggttctctctatgcctcaagtgaatattgtcaagaagactgaagtccagacgggcgggttctcaaaggaatctattctgcccaagcggaactcggataagcttatcgccagaaagaaggactgggacccgaagaagtatggaggtttcgactcaccaacggtggcttactctgtcctggttgtggcaaaggtggagaagggaaagtcaaagaagctcaagtctgtcaaggagctcctgggtatcaccattatggagaggtccagcttcgaaaagaatccgatcgattttctcgaggcgaagggatataaggaagtgaagaaggacctgatcattaagcttccaaagtacagtcttttcgagttggaaaacggcaggaagcgcatgttggcttccgcaggagagctccagaagggtaacgagcttgctttgccgtccaagtatgtgaacttcctctatctggcatcccactacgagaagctcaagggcagcccagaggataacgaacagaagcaactgtttgtggagcaacacaagcattatcttgacgagatcattgaacagatttcggagttcagtaagcgcgtcatcctcgccgacgcgaatttggataaggttctctcagcctacaacaagcaccgggacaagcctatcagagagcaggcggaaaatatcattcatctcttcaccctgacaaaccttggggctcccgctgcattcaagtattttgacactacgattgatcggaagagatacacttctacgaaggaggtgctggatgcaacccttatccaccaatcgattactggcctctacgagacgcggatcgacttgagtcagctcgggggggataagagaccagcggcaaccaagaaggcaggacaagcgaagaagaagaagtagSpCas9 amino acid sequence SEQ ID NO: 3MAPKKKRKVGIHGVPAAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHANDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK eSpCas9(1.0) amino acid sequence SEQ ID NO: 4MAPKKKRKVGIHGVPAAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEALYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHANDAYLNAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK eSpCas9(1.1) amino acid sequence SEQ ID NO: 5MAPKKKRKVGIHGVPAAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHANDAYLNAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ1SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK SpCas9-HF1 amino acid sequence SEQ ID NO: 6MAPKKKRKVGIHGVPAAMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHANDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK eSpCas9(1.0) codon-optimized nucleotide sequenceSEQ ID NO: 7atggcccctaagaagaagagaaaggtcggtattcacggcgttcctgcggcgatggacaagaagtatagtattggtctggacattgggacgaattccgttggctgggccgtgatcaccgatgagtacaaggtcccttccaagaagtttaaggttctggggaacaccgatcggcacagcatcaagaagaatctcattggagccctcctgttcgactcaggcgagaccgccgaagcaacaaggctcaagagaaccgcaaggagacggtatacaagaaggaagaataggatctgctacctgcaggagattttcagcaacgaaatggcgaaggtggacgattcgttctttcatagattggaggagagtttcctcgtcgaggaagataagaagcacgagaggcatcctatctttggcaacattgtcgacgaggttgcctatcacgaaaagtaccccacaatctatcatctgcggaagaagcttgtggactcgactgataaggcggaccttagattgatctacctcgctctggcacacatgattaagttcaggggccattttctgatcgagggggatcttaacccggacaatagcgatgtggacaagttgttcatccagctcgtccaaacctacaatcagctctttgaggaaaacccaattaatgcttcaggcgtcgacgccaaggcgatcctgtctgcacgcctttcaaagtctcgccggcttgagaacttgatcgctcaactcccgggcgaaaagaagaacggcttgttcgggaatctcattgcactttcgttggggctcacaccaaacttcaagagtaattttgatctcgctgaggacgcaaagctgcagctttccaaggacacttatgacgatgacctggataaccttttggcccaaatcggcgatcagtacgcggacttgttcctcgccgcgaagaatttgtcggacgcgatcctcctgagtgatattctccgcgtgaacaccgagattacaaaggccccgctctcggcgagtatgatcaagcgctatgacgagcaccatcaggatctgacccttttgaaggctttggtccggcagcaactcccagagaagtacaaggaaatcttctttgatcaatccaagaacggctacgctggttatattgacggcggggcatcgcaggaggaattctacaagtttatcaagccaattctggagaagatggatggcacagaggaactcctggtgaagctcaatagggaggaccttttgcggaagcaaagaactttcgataacggcagcatccctcaccagattcatctcggggagctgcacgccatcctgagaaggcaggaagacttctacccctttcttaaggataaccgggagaagatcgaaaagattctgacgttcagaattccgtactatgtcggaccactcgcccggggtaattccagatttgcgtggatgaccagaaagagcgaggaaaccatcacaccttggaacttcgaggaagtggtcgataagggcgcttccgcacagagcttcattgagcgcatgacaaattttgacaagaacctgcctaatgagaaggtccttcccaagcattccctcctgtacgagtatttcactgtttataacgaactcacgaaggtgaagtatgtgaccgagggaatgcgcaagcccgccttcctgagcggcgagcaaaagaaggcgatcgtggaccttttgtttaagaccaatcggaaggtcacagttaagcagctcaaggaggactacttcaagaagattgaatgcttcgattccgttgagatcagcggcgtggaagacaggtttaacgcgtcactggggacttaccacgatctcctgaagatcattaaggataaggacttcttggacaacgaggaaaatgaggatatcctcgaagacattgtcctgactcttacgttgtttgaggatagggaaatgatcgaggaacgcttgaagacgtatgcccatctcttcgatgacaaggttatgaagcagctcaagagaagaagatacaccggatggggaaggctgtcccgcaagcttatcaatggcattagagacaagcaatcagggaagacaatccttgactttttgaagtctgatggcttcgcgaacaggaattttatgcagctgattcacgatgactcacttactttcaaggaggatatccagaaggctcaagtgtcgggacaaggtgacagtctgcacgagcatatcgccaaccttgcgggatctcctgcaatcaagaagggtattctgcagacagtcaaggttgtggatgagcttgtgaaggtcatgggacggcataagcccgagaacatcgttattgagatggccagagaaaatcagaccacacaaaagggtcagaagaactcgagggagcgcatgaagcgcatcgaggaaggcattaaggagctggggagtcagatccttaaggagcacccggtggaaaacacgcagttgcaaaatgaggccctctatctgtactatctgcaaaatggcagggatatgtatgtggaccaggagttggatattaaccgcctctcggattacgacgtcgatcatatcgttcctcagtccttccttaaggatgacagcattgacaataaggttctcaccaggtccgacaagaaccgcgggaagtccgataatgtgcccagcgaggaagtcgttaagaagatgaagaactactggaggcaacttttgaatgccaagttgatcacacagaggaagtttgataacctcactaaggccgagcgcggaggtctcagcgaactggacaaggcgggcttcattaagcggcaactggttgagactagacagatcacgaagcacgtggcgcagattctcgattcacgcatgaacacgaagtacgatgagaatgacaagctgatccgggaagtgaaggtcatcaccttgaagtcaaagctcgtttctgacttcaggaaggatttccaattttataaggtgcgcgagatcaacaattatcaccatgctcatgacgcatacctcaacgctgtggtcggaacagcattgattaagaagtacccggcgctcgagtccgaattcgtgtacggtgactataaggtttacgatgtgcgcaagatgatcgccaagtcagagcaggaaattggcaaggccactgcgaagtatttcttttactctaacattatgaatttctttaagactgagatcacgctggctaatggcgaaatccggaaggcgccacttattgagaccaacggcgagacaggggaaatcgtgtgggacaaggggagggatttcgccacagtccgcaaggttctctctatgcctcaagtgaatattgtcaagaagactgaagtccagacgggcgggttctcaaaggaatctattctgcccaagcggaactcggataagcttatcgccagaaagaaggactgggacccgaagaagtatggaggtttcgactcaccaacggtggcttactctgtcctggttgtggcaaaggtggagaagggaaagtcaaagaagctcaagtctgtcaaggagctcctgggtatcaccattatggagaggtccagcttcgaaaagaatccgatcgattttctcgaggcgaagggatataaggaagtgaagaaggacctgatcattaagcttccaaagtacagtcttttcgagttggaaaacggcaggaagcgcatgttggcttccgcaggagagctccagaagggtaacgagcttgctttgccgtccaagtatgtgaacttcctctatctggcatcccactacgagaagctcaagggcagcccagaggataacgaacagaagcaactgtttgtggagcaacacaagcattatcttgacgagatcattgaacagatttcggagttcagtaagcgcgtcatcctcgccgacgcgaatttggataaggttctctcagcctacaacaagcaccgggacaagcctatcagagagcaggcggaaaatatcattcatctcttcaccctgacaaaccttggggctcccgctgcattcaagtattttgacactacgattgatcggaagagatacacttctacgaaggaggtgctggatgcaacccttatccaccaatcgattactggcctctacgagacgcggatcgacttgagtcagctcgggggggataagagaccagcggcaaccaagaaggcaggacaagcgaagaagaagaagtageSpCas9(1.1) codon-optimized nucleotide sequence SEQ ID NO: 8atggcccctaagaagaagagaaaggtcggtattcacggcgttcctgcggcgatggacaagaagtatagtattggtctggacattgggacgaattccgttggctgggccgtgatcaccgatgagtacaaggtcccttccaagaagtttaaggttctggggaacaccgatcggcacagcatcaagaagaatctcattggagccctcctgttcgactcaggcgagaccgccgaagcaacaaggctcaagagaaccgcaaggagacggtatacaagaaggaagaataggatctgctacctgcaggagattttcagcaacgaaatggcgaaggtggacgattcgttctttcatagattggaggagagtttcctcgtcgaggaagataagaagcacgagaggcatcctatctttggcaacattgtcgacgaggttgcctatcacgaaaagtaccccacaatctatcatctgcggaagaagcttgtggactcgactgataaggcggaccttagattgatctacctcgctctggcacacatgattaagttcaggggccattttctgatcgagggggatcttaacccggacaatagcgatgtggacaagttgttcatccagctcgtccaaacctacaatcagctctttgaggaaaacccaattaatgcttcaggcgtcgacgccaaggcgatcctgtctgcacgcctttcaaagtctcgccggcttgagaacttgatcgctcaactcccgggcgaaaagaagaacggcttgttcgggaatctcattgcactttcgttggggctcacaccaaacttcaagagtaattttgatctcgctgaggacgcaaagctgcagctttccaaggacacttatgacgatgacctggataaccttttggcccaaatcggcgatcagtacgcggacttgttcctcgccgcgaagaatttgtcggacgcgatcctcctgagtgatattctccgcgtgaacaccgagattacaaaggccccgctctcggcgagtatgatcaagcgctatgacgagcaccatcaggatctgacccttttgaaggctttggtccggcagcaactcccagagaagtacaaggaaatcttctttgatcaatccaagaacggctacgctggttatattgacggcggggcatcgcaggaggaattctacaagtttatcaagccaattctggagaagatggatggcacagaggaactcctggtgaagctcaatagggaggaccttttgcggaagcaaagaactttcgataacggcagcatccctcaccagattcatctcggggagctgcacgccatcctgagaaggcaggaagacttctacccctttcttaaggataaccgggagaagatcgaaaagattctgacgttcagaattccgtactatgtcggaccactcgcccggggtaattccagatttgcgtggatgaccagaaagagcgaggaaaccatcacaccttggaacttcgaggaagtggtcgataagggcgcttccgcacagagcttcattgagcgcatgacaaattttgacaagaacctgcctaatgagaaggtccttcccaagcattccctcctgtacgagtatttcactgtttataacgaactcacgaaggtgaagtatgtgaccgagggaatgcgcaagcccgccttcctgagcggcgagcaaaagaaggcgatcgtggaccttttgtttaagaccaatcggaaggtcacagttaagcagctcaaggaggactacttcaagaagattgaatgcttcgattccgttgagatcagcggcgtggaagacaggtttaacgcgtcactggggacttaccacgatctcctgaagatcattaaggataaggacttcttggacaacgaggaaaatgaggatatcctcgaagacattgtcctgactcttacgttgtttgaggatagggaaatgatcgaggaacgcttgaagacgtatgcccatctcttcgatgacaaggttatgaagcagctcaagagaagaagatacaccggatggggaaggctgtcccgcaagcttatcaatggcattagagacaagcaatcagggaagacaatccttgactttttgaagtctgatggcttcgcgaacaggaattttatgcagctgattcacgatgactcacttactttcaaggaggatatccagaaggctcaagtgtcgggacaaggtgacagtctgcacgagcatatcgccaaccttgcgggatctcctgcaatcaagaagggtattctgcagacagtcaaggttgtggatgagcttgtgaaggtcatgggacggcataagcccgagaacatcgttattgagatggccagagaaaatcagaccacacaaaagggtcagaagaactcgagggagcgcatgaagcgcatcgaggaaggcattaaggagctggggagtcagatccttaaggagcacccggtggaaaacacgcagttgcaaaatgagaagctctatctgtactatctgcaaaatggcagggatatgtatgtggaccaggagttggatattaaccgcctctcggattacgacgtcgatcatatcgttcctcagtccttccttgcggatgacagcattgacaataaggttctcaccaggtccgacaagaaccgcgggaagtccgataatgtgcccagcgaggaagtcgttaagaagatgaagaactactggaggcaacttttgaatgccaagttgatcacacagaggaagtttgataacctcactaaggccgagcgcggaggtctcagcgaactggacaaggcgggcttcattaagcggcaactggttgagactagacagatcacgaagcacgtggcgcagattctcgattcacgcatgaacacgaagtacgatgagaatgacaagctgatccgggaagtgaaggtcatcaccttgaagtcaaagctcgtttctgacttcaggaaggatttccaattttataaggtgcgcgagatcaacaattatcaccatgctcatgacgcatacctcaacgctgtggtcggaacagcattgattaagaagtacccggcgctcgagtccgaattcgtgtacggtgactataaggtttacgatgtgcgcaagatgatcgccaagtcagagcaggaaattggcaaggccactgcgaagtatttcttttactctaacattatgaatttctttaagactgagatcacgctggctaatggcgaaatccggaaggcgccacttattgagaccaacggcgagacaggggaaatcgtgtgggacaaggggagggatttcgccacagtccgcaaggttctctctatgcctcaagtgaatattgtcaagaagactgaagtccagacgggcgggttctcaaaggaatctattctgcccaagcggaactcggataagcttatcgccagaaagaaggactgggacccgaagaagtatggaggtttcgactcaccaacggtggcttactctgtcctggttgtggcaaaggtggagaagggaaagtcaaagaagctcaagtctgtcaaggagctcctgggtatcaccattatggagaggtccagcttcgaaaagaatccgatcgattttctcgaggcgaagggatataaggaagtgaagaaggacctgatcattaagcttccaaagtacagtcttttcgagttggaaaacggcaggaagcgcatgttggcttccgcaggagagctccagaagggtaacgagcttgctttgccgtccaagtatgtgaacttcctctatctggcatcccactacgagaagctcaagggcagcccagaggataacgaacagaagcaactgtttgtggagcaacacaagcattatcttgacgagatcattgaacagatttcggagttcagtaagcgcgtcatcctcgccgacgcgaatttggataaggttctctcagcctacaacaagcaccgggacaagcctatcagagagcaggcggaaaatatcattcatctcttcaccctgacaaaccttggggctcccgctgcattcaagtattttgacactacgattgatcggaagagatacacttctacgaaggaggtgctggatgcaacccttatccaccaatcgattactggcctctacgagacgcggatcgacttgagtcagctcgggggggataagagaccagcggcaaccaagaaggcaggacaagcgaagaagaagaagtagSpCas9-HF1 codon-optimized nucleotide sequence SEQ ID NO: 9atggcccctaagaagaagagaaaggtcggtattcacggcgttcctgcggcgatggacaagaagtatagtattggtctggacattgggacgaattccgttggctgggccgtgatcaccgatgagtacaaggtcccttccaagaagtttaaggttctggggaacaccgatcggcacagcatcaagaagaatctcattggagccctcctgttcgactcaggcgagaccgccgaagcaacaaggctcaagagaaccgcaaggagacggtatacaagaaggaagaataggatctgctacctgcaggagattttcagcaacgaaatggcgaaggtggacgattcgttctttcatagattggaggagagtttcctcgtcgaggaagataagaagcacgagaggcatcctatctttggcaacattgtcgacgaggttgcctatcacgaaaagtaccccacaatctatcatctgcggaagaagcttgtggactcgactgataaggcggaccttagattgatctacctcgctctggcacacatgattaagttcaggggccattttctgatcgagggggatcttaacccggacaatagcgatgtggacaagttgttcatccagctcgtccaaacctacaatcagctctttgaggaaaacccaattaatgcttcaggcgtcgacgccaaggcgatcctgtctgcacgcctttcaaagtctcgccggcttgagaacttgatcgctcaactcccgggcgaaaagaagaacggcttgttcgggaatctcattgcactttcgttggggctcacaccaaacttcaagagtaattttgatctcgctgaggacgcaaagctgcagctttccaaggacacttatgacgatgacctggataaccttttggcccaaatcggcgatcagtacgcggacttgttcctcgccgcgaagaatttgtcggacgcgatcctcctgagtgatattctccgcgtgaacaccgagattacaaaggccccgctctcggcgagtatgatcaagcgctatgacgagcaccatcaggatctgacccttttgaaggctttggtccggcagcaactcccagagaagtacaaggaaatcttctttgatcaatccaagaacggctacgctggttatattgacggcggggcatcgcaggaggaattctacaagtttatcaagccaattctggagaagatggatggcacagaggaactcctggtgaagctcaatagggaggaccttttgcggaagcaaagaactttcgataacggcagcatccctcaccagattcatctcggggagctgcacgccatcctgagaaggcaggaagacttctacccctttcttaaggataaccgggagaagatcgaaaagattctgacgttcagaattccgtactatgtcggaccactcgcccggggtaattccagatttgcgtggatgaccagaaagagcgaggaaaccatcacaccttggaacttcgaggaagtggtcgataagggcgcttccgcacagagcttcattgagcgcatgacaGCCtttgacaagaacctgcctaatgagaaggtccttcccaagcattccctcctgtacgagtatttcactgtttataacgaactcacgaaggtgaagtatgtgaccgagggaatgcgcaagcccgccttcctgagcggcgagcaaaagaaggcgatcgtggaccttttgtttaagaccaatcggaaggtcacagttaagcagctcaaggaggactacttcaagaagattgaatgcttcgattccgttgagatcagcggcgtggaagacaggtttaacgcgtcactggggacttaccacgatctcctgaagatcattaaggataaggacttcttggacaacgaggaaaatgaggatatcctcgaagacattgtcctgactcttacgttgtttgaggatagggaaatgatcgaggaacgcttgaagacgtatgcccatctcttcgatgacaaggttatgaagcagctcaagagaagaagatacaccggatggggaGCCctgtcccgcaagcttatcaatggcattagagacaagcaatcagggaagacaatccttgactttttgaagtctgatggcttcgcgaacaggaattttatgGCCctgattcacgatgactcacttactttcaaggaggatatccagaaggctcaagtgtcgggacaaggtgacagtctgcacgagcatatcgccaaccttgcgggatctcctgcaatcaagaagggtattctgcagacagtcaaggttgtggatgagcttgtgaaggtcatgggacggcataagcccgagaacatcgttattgagatggccagagaaaatcagaccacacaaaagggtcagaagaactcgagggagcgcatgaagcgcatcgaggaaggcattaaggagctggggagtcagatccttaaggagcacccggtggaaaacacgcagttgcaaaatgagaagctctatctgtactatctgcaaaatggcagggatatgtatgtggaccaggagttggatattaaccgcctctcggattacgacgtcgatcatatcgttcctcagtccttccttaaggatgacagcattgacaataaggttctcaccaggtccgacaagaaccgcgggaagtccgataatgtgcccagcgaggaagtcgttaagaagatgaagaactactggaggcaacttttgaatgccaagttgatcacacagaggaagtttgataacctcactaaggccgagcgcggaggtctcagcgaactggacaaggcgggcttcattaagcggcaactggttgagactagaGCCatcacgaagcacgtggcgcagattctcgattcacgcatgaacacgaagtacgatgagaatgacaagctgatccgggaagtgaaggtcatcaccttgaagtcaaagctcgtttctgacttcaggaaggatttccaattttataaggtgcgcgagatcaacaattatcaccatgctcatgacgcatacctcaacgctgtggtcggaacagcattgattaagaagtacccgaagctcgagtccgaattcgtgtacggtgactataaggtttacgatgtgcgcaagatgatcgccaagtcagagcaggaaattggcaaggccactgcgaagtatttcttttactctaacattatgaatttctttaagactgagatcacgctggctaatggcgaaatccggaagagaccacttattgagaccaacggcgagacaggggaaatcgtgtgggacaaggggagggatttcgccacagtccgcaaggttctctctatgcctcaagtgaatattgtcaagaagactgaagtccagacgggcgggttctcaaaggaatctattctgcccaagcggaactcggataagcttatcgccagaaagaaggactgggacccgaagaagtatggaggtttcgactcaccaacggtggcttactctgtcctggttgtggcaaaggtggagaagggaaagtcaaagaagctcaagtctgtcaaggagctcctgggtatcaccattatggagaggtccagcttcgaaaagaatccgatcgattttctcgaggcgaagggatataaggaagtgaagaaggacctgatcattaagcttccaaagtacagtcttttcgagttggaaaacggcaggaagcgcatgttggcttccgcaggagagctccagaagggtaacgagcttgctttgccgtccaagtatgtgaacttcctctatctggcatcccactacgagaagctcaagggcagcccagaggataacgaacagaagcaactgtttgtggagcaacacaagcattatcttgacgagatcattgaacagatttcggagttcagtaagcgcgtcatcctcgccgacgcgaatttggataaggttctctcagcctacaacaagcaccgggacaagcctatcagagagcaggcggaaaatatcattcatctcttcaccctgacaaaccttggggctcccgctgcattcaagtattttgacactacgattgatcggaagagatacacttctacgaaggaggtgctggatgcaacccttatccaccaatcgattactggcctctacgagacgcggatcgacttgagtcagctcgggggggataagagaccagcggcaaccaagaaggcaggacaagcgaagaagaagaagtagpJIT163-SpCas9 vector sequence SEQ ID NO: 10gagctcggtacctgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgatcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcaaagcttccaccatggcgtgcaggtcgactctagaggatccccatggcccctaagaagaagagaaaggtcggtattcacggcgttcctgcggcgatggacaagaagtatagtattggtctggacattgggacgaattccgttggctgggccgtgatcaccgatgagtacaaggtcccttccaagaagtttaaggttctggggaacaccgatcggcacagcatcaagaagaatctcattggagccctcctgttcgactcaggcgagaccgccgaagcaacaaggctcaagagaaccgcaaggagacggtatacaagaaggaagaataggatctgctacctgcaggagattttcagcaacgaaatggcgaaggtggacgattcgttctttcatagattggaggagagtttcctcgtcgaggaagataagaagcacgagaggcatcctatctttggcaacattgtcgacgaggttgcctatcacgaaaagtaccccacaatctatcatctgcggaagaagcttgtggactcgactgataaggcggaccttagattgatctacctcgctctggcacacatgattaagttcaggggccattttctgatcgagggggatcttaacccggacaatagcgatgtggacaagttgttcatccagctcgtccaaacctacaatcagctctttgaggaaaacccaattaatgcttcaggcgtcgacgccaaggcgatcctgtctgcacgcctttcaaagtctcgccggcttgagaacttgatcgctcaactcccgggcgaaaagaagaacggcttgttcgggaatctcattgcactttcgttggggctcacaccaaacttcaagagtaattttgatctcgctgaggacgcaaagctgcagctttccaaggacacttatgacgatgacctggataaccttttggcccaaatcggcgatcagtacgcggacttgttcctcgccgcgaagaatttgtcggacgcgatcctcctgagtgatattctccgcgtgaacaccgagattacaaaggccccgctctcggcgagtatgatcaagcgctatgacgagcaccatcaggatctgacccttttgaaggctttggtccggcagcaactcccagagaagtacaaggaaatcttctttgatcaatccaagaacggctacgctggttatattgacggcggggcatcgcaggaggaattctacaagtttatcaagccaattctggagaagatggatggcacagaggaactcctggtgaagctcaatagggaggaccttttgcggaagcaaagaactttcgataacggcagcatccctcaccagattcatctcggggagctgcacgccatcctgagaaggcaggaagacttctacccctttcttaaggataaccgggagaagatcgaaaagattctgacgttcagaattccgtactatgtcggaccactcgcccggggtaattccagatttgcgtggatgaccagaaagagcgaggaaaccatcacaccttggaacttcgaggaagtggtcgataagggcgcttccgcacagagcttcattgagcgcatgacaaattttgacaagaacctgcctaatgagaaggtccttcccaagcattccctcctgtacgagtatttcactgtttataacgaactcacgaaggtgaagtatgtgaccgagggaatgcgcaagcccgccttcctgagcggcgagcaaaagaaggcgatcgtggaccttttgtttaagaccaatcggaaggtcacagttaagcagctcaaggaggactacttcaagaagattgaatgcttcgattccgttgagatcagcggcgtggaagacaggtttaacgcgtcactggggacttaccacgatctcctgaagatcattaaggataaggacttcttggacaacgaggaaaatgaggatatcctcgaagacattgtcctgactcttacgttgtttgaggatagggaaatgatcgaggaacgcttgaagacgtatgcccatctcttcgatgacaaggttatgaagcagctcaagagaagaagatacaccggatggggaaggctgtcccgcaagcttatcaatggcattagagacaagcaatcagggaagacaatccttgactttttgaagtctgatggcttcgcgaacaggaattttatgcagctgattcacgatgactcacttactttcaaggaggatatccagaaggctcaagtgtcgggacaaggtgacagtctgcacgagcatatcgccaaccttgcgggatctcctgcaatcaagaagggtattctgcagacagtcaaggttgtggatgagcttgtgaaggtcatgggacggcataagcccgagaacatcgttattgagatggccagagaaaatcagaccacacaaaagggtcagaagaactcgagggagcgcatgaagcgcatcgaggaaggcattaaggagctggggagtcagatccttaaggagcacccggtggaaaacacgcagttgcaaaatgagaagctctatctgtactatctgcaaaatggcagggatatgtatgtggaccaggagttggatattaaccgcctctcggattacgacgtcgatcatatcgttcctcagtccttccttaaggatgacagcattgacaataaggttctcaccaggtccgacaagaaccgcgggaagtccgataatgtgcccagcgaggaagtcgttaagaagatgaagaactactggaggcaacttttgaatgccaagttgatcacacagaggaagtttgataacctcactaaggccgagcgcggaggtctcagcgaactggacaaggcgggcttcattaagcggcaactggttgagactagacagatcacgaagcacgtggcgcagattctcgattcacgcatgaacacgaagtacgatgagaatgacaagctgatccgggaagtgaaggtcatcaccttgaagtcaaagctcgtttctgacttcaggaaggatttccaattttataaggtgcgcgagatcaacaattatcaccatgctcatgacgcatacctcaacgctgtggtcggaacagcattgattaagaagtacccgaagctcgagtccgaattcgtgtacggtgactataaggtttacgatgtgcgcaagatgatcgccaagtcagagcaggaaattggcaaggccactgcgaagtatttcttttactctaacattatgaatttctttaagactgagatcacgctggctaatggcgaaatccggaagagaccacttattgagaccaacggcgagacaggggaaatcgtgtgggacaaggggagggatttcgccacagtccgcaaggttctctctatgcctcaagtgaatattgtcaagaagactgaagtccagacgggcgggttctcaaaggaatctattctgcccaagcggaactcggataagcttatcgccagaaagaaggactgggacccgaagaagtatggaggtttcgactcaccaacggtggcttactctgtcctggttgtggcaaaggtggagaagggaaagtcaaagaagctcaagtctgtcaaggagctcctgggtatcaccattatggagaggtccagcttcgaaaagaatccgatcgattttctcgaggcgaagggatataaggaagtgaagaaggacctgatcattaagcttccaaagtacagtcttttcgagttggaaaacggcaggaagcgcatgttggcttccgcaggagagctccagaagggtaacgagcttgctttgccgtccaagtatgtgaacttcctctatctggcatcccactacgagaagctcaagggcagcccagaggataacgaacagaagcaactgtttgtggagcaacacaagcattatcttgacgagatcattgaacagatttcggagttcagtaagcgcgtcatcctcgccgacgcgaatttggataaggttctctcagcctacaacaagcaccgggacaagcctatcagagagcaggcggaaaatatcattcatctcttcaccctgacaaaccttggggctcccgctgcattcaagtattttgacactacgattgatcggaagagatacacttctacgaaggaggtgctggatgcaacccttatccaccaatcgattactggcctctacgagacgcggatcgacttgagtcagctcgggggggataagagaccagcggcaaccaagaaggcaggacaagcgaagaagaagaagtaggggcgagctcgaattcgctgaaatcaccagtctctctctacaaatctatctctctctattttctccataaataatgtgtgagtagtttcccgataagggaaattagggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatctcctaaagtccctatagatctttgtcgtgaatataaaccagacacgagacgactaaacctggagcccagacgccgttcgaagctagaagtaccgcttaggcaggaggccgttagggaaaagatgctaaggcagggttggttacgttgactcccccgtaggtttggtttaaatatgatgaagtggacggaaggaaggaggaagacaaggaaggataaggttgcaggccctgtgcaaggtaagaagatggaaatttgatagaggtacgctactatacttatactatacgctaagggaatgcttgtatttataccctataccccctaataaccccttatcaatttaagaaataatccgcataagcccccgcttaaaaattggtatcagagccatgaataggtctatgaccaaaactcaagaggataaaacctcaccaaaatacgaaagagttcttaactctaaagataaaagatctttcaagatcaaaactagttccctcacaccggagcatgcgatatcctcgagagatctaggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgt pUC57-U3-tRNA-sgRNA vector sequence SEQ ID NO: 11tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccagatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgcctgcaggtcgacgattaaggaatctttaaacatacgaacagatcacttaaagttcttctgaagcaacttaaagttatcaggcatgcatggatcttggaggaatcagatgtgcagtcagggaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccgggaacactgggtacgtcggaaaccacgtgatgtgaagaagtaagataaactgtaggagaaaagcatttcgtagtgggccatgaagcctttcaggacatgtattgcagtatgggccggcccattacgcaattggacgacaacaaagactagtattagtaccacctcggctatccacatagatcaaagctgatttaaaagagttgtgcagatgatccgtggcaacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgcaagagaccgatatcccatggctcgagggtctcggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttccacataatctctagaggatccccggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgactcccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc sequence of 5′ end ribozyme SEQ ID NO: 12NNNNNNCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC sequence of 3′ end ribozymeSEQ ID NO: 13GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACSEQ ID NO: 14 AAGAAGAGAAAGGTC SEQ ID NO: 15 CCCAAGAAGAAGAGGAAGGTGSEQ ID NO: 16 CCAAAGAAGAAGAGGAAGGTT SEQ ID NO: 17 SGGSPKKKRKVSEQ ID NO: 18 TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG SEQ ID NO: 19 PKKKRKVSEQ ID NO: 20 AGGTCGGGGAGGGGACGTACGGG SEQ ID NO: 21GGCAAGGTCGGGGAGGGGACGTAC SEQ ID NO: 22 AAACGTACGTCCCCTCCCCGACCTSEQ ID NO: 23 GACGTCGGCGAGGAAGGCCTCGG SEQ ID NO: 24GGCAGACGTCGGCGAGGAAGGCCT SEQ ID NO: 25 AAACAGGCCTTCCTCGCCGACGTCSEQ ID NO: 26 CATGGTGGGGAAAGCTTGGAGGG SEQ ID NO: 27GGCACATGGTGGGGAAAGCTTGGA SEQ ID NO: 28 AAACTCCAAGCTTTCCCCACCATGSEQ ID NO: 29 CCGGACGACGACGTCGACGACGG SEQ ID NO: 30GGCACCGGACGACGACGTCGACGA SEQ ID NO: 31 AAACTCGTCGACGTCGTCGTCCGGSEQ ID NO: 32 TTGAAGTCCCTTCTAGATGGAGG SEQ ID NO: 33GGCATTGAAGTCCCTTCTAGATGG SEQ ID NO: 34 AAACCCATCTAGAAGGGACTTCAASEQ ID NO: 35 ACTGCGACACCCAGATATCGTGG SEQ ID NO: 36GGCAACTGCGACACCCAGATATCG SEQ ID NO: 37 AAACCGATATCTGGGTGTCGCAGTSEQ ID NO: 38 GTTGGTCTTTGCTCCTGCAGAGG SEQ ID NO: 39GGCAGTTGGTCTTTGCTCCTGCAG SEQ ID NO: 40 AAACCTGCAGGAGCAAAGACCAACSEQ ID NO: 41 TGCAAGGTCGGGGAGGGGACGTAC SEQ ID NO: 42TGCAGACGTCGGCGAGGAAGGCCT SEQ ID NO: 43 TGCACATGGTGGGGAAAGCTTGGASEQ ID NO: 44 TGCACCGGACGACGACGTCGACGA SEQ ID NO: 45TGCATTGAAGTCCCTTCTAGATGG SEQ ID NO: 46 TGCACTGCGACACCCAGATATCGSEQ ID NO: 47 TGCAGTTGGTCTTTGCTCCTGCAG SEQ ID NO: 48GGCGTTGGTCTTTGCTCCTGCAG SEQ ID NO: 49 GGCGGTTGGTCTTTGCTCCTGCAGSEQ ID NO: 50 GGTGAGTGAGTGTGTGCGTGTGG SEQ ID NO: 51CACCGGTGAGTGAGTGTGTGCGTG SEQ ID NO: 52 AAACCACGCACACACTCACTCACCSEQ ID NO: 53 CACCGGGTGAGTGAGTGTGTGCGTG SEQ ID NO: 54AAACCACGCACACACTCACTCACCC SEQ ID NO: 55CACCGaacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgcaGGTGAGTGAGTGTGTGCGTG SEQ ID NO: 56AAACCACGCACACACTCACTCACCtgcaccagccgggaatcgaacccgggtctgtaccgtggcagggtactattctaccactagaccactggtgctttgttC SEQ ID NO: 57 NNNNNNNNNNNNNNNNNNNNTTTTTTTSEQ ID NO: 58 NNNNNNNNNNNNNNNNNNNTTTTTTT SEQ ID NO: 59TGGAGTTGGTCTTTGCTCCTGCAGAGG SEQ ID NO: 60 GACGCCGGCGAGGAAGGCCTCGGSEQ ID NO: 61 GCAGTCGGAGAGGAAGGCCTGGG SEQ ID NO: 62AGATCGGGGAGGGGACGTACGGG SEQ ID NO: 63 AGGTGGGGGAAGGGACGTACGGGSEQ ID NO: 64 AGATTGGGGAGGGCACGTACGGG SEQ ID NO: 65AGCGTCGGCGAGGAAGGCCTCGG SEQ ID NO: 66 GGTGTCGGCGAGGAAGGCCTCGGSEQ ID NO: 67 GATATCGGCGAGGAAGGCCTCGG SEQ ID NO: 68GACACCGGCGAGGAAGGCCTCGG SEQ ID NO: 69 GACGCTGGCGAGGAAGGCCTCGGSEQ ID NO: 70 GACGTTAGCGAGGAAGGCCTCGG SEQ ID NO: 71GACGTCAACGAGGAAGGCCTCGG SEQ ID NO: 72 GACGTCGATGAGGAAGGCCTCGGSEQ ID NO: 73 GACGTCGGTAAGGAAGGCCTCGG SEQ ID NO: 74GACGTCGGCAGGGAAGGCCTCGG SEQ ID NO: 75 GACGTCGGCGGAGAAGGCCTCGGSEQ ID NO: 76 GACGTCGGCGAAAAAGGCCTCGG SEQ ID NO: 77GACGTCGGCGAGAGAGGCCTCGG SEQ ID NO: 78 GACGTCGGCGAGGGGGGCCTCGGSEQ ID NO: 79 GACGTCGGCGAGGAGAGCCTCGG SEQ ID NO: 80GACGTCGGCGAGGAAAACCTCGG SEQ ID NO: 81 GACGTCGGCGAGGAAGATCTCGGSEQ ID NO: 82 GACGTCGGCGAGGAAGGTTTCGG SEQ ID NO: 83GACGTCGGCGAGGAAGGCTCCGG SEQ ID NO: 84 GCTGAGTGAGTGTATGCGTGTGGSEQ ID NO: 85 TGTGGGTGAGTGTGTGCGTGAGG

1. A genome editing system for site-directed modification of a targetsequence in the genome of a cell, which comprises at least one selectedfrom the following i) to iii): i) a Cas9 nuclease variant, and anexpression construct comprising a nucleotide sequence encoding atRNA-guide RNA fusion; ii) an expression construct comprising anucleotide sequence encoding a Cas9 nuclease variant, and an expressionconstruct comprising a nucleotide sequence encoding a tRNA-guide RNAfusion; and iii) an expression construct comprising a nucleotidesequence encoding a Cas9 nuclease variant and a nucleotide sequenceencoding a tRNA-guide RNA fusion; wherein the Cas9 nuclease variant hashigher specificity as compared with the wild-type Cas9 nuclease, whereinthe 5′ end of the guide RNA is linked to the 3′ end of the tRNA, whereinthe fusion is cleaved at the 5′ end of the guide RNA after beingtranscribed in the cell, thereby forming a guide RNA that does not carryan extra nucleotide at the 5′ end.
 2. A genome editing system forsite-directed modification of a target sequence in the genome of a cell,which comprises at least one selected from the following i) to iii): i)a Cas9 nuclease variant, and an expression construct comprising anucleotide sequence encoding a ribozyme-guide RNA fusion; ii) anexpression construct comprising a nucleotide sequence encoding a Cas9nuclease variant, and an expression construct comprising a nucleotidesequence encoding a ribozyme-guide RNA fusion; and iii) an expressionconstruct comprising a nucleotide sequence encoding a Cas9 nucleasevariant and a nucleotide sequence encoding a ribozyme-guide RNA fusion;wherein the Cas9 nuclease variant has higher specificity as comparedwith the wild-type Cas9 nuclease, wherein the 5′ end of the guide RNA islinked to the 3′ end of a first ribozyme, wherein the first ribozyme isdesigned to cleave the fusion at the 5′ end of the guide RNA, therebyforming a guide RNA that does not carry extra nucleotide at the 5′ end.3. The system of claim 1, wherein the tRNA and the cell to be modifiedare derived from a same species.
 4. The system of claim 1, wherein thetRNA is encoded by a sequence as shown in SEQ ID NO:1.
 5. The system ofclaim 1, wherein the Cas9 nuclease variant is a variant of SEQ ID NO:2and comprises an amino acid substitution at position 855 of SEQ ID NO:2,for example, the amino acid substitution is K855A.
 6. The system ofclaim 1, wherein the Cas9 nuclease variant is a variant of the SEQ IDNO:2 and comprises amino acid substitutions at positions 810, 1003 and1060 of SEQ ID NO:2, for example, the amino acid substitutions areK810A, K1003A and R1060A.
 7. The system of claim 1, wherein the Cas9nuclease variant is a variant of the SEQ ID NO:2 and comprises aminoacid substitutions at positions 848, 1003 and 1060 of SEQ ID NO:2, forexample, the amino acid substitutions are K848A, K1003A and R1060A. 8.The system of claim 1, wherein the Cas9 nuclease variant is a variant ofthe SEQ ID NO:2 and comprises amino acid substitutions at positions 611,695 and 926 of SEQ ID NO:2, for example, the amino acid substitutionsare R611A, Q695A and Q926A.
 9. The system of claim 1, wherein the Cas9nuclease variant is a variant of the SEQ ID NO:2 and comprises aminoacid substitutions at positions 497, 611, 695 and 926 of SEQ ID NO:2,for example, the amino acid substitutions are N497A, R611A, Q695A andQ926A.
 10. The system of claim 1, wherein the Cas9 nuclease variantcomprises an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6.
 11. The system of claim 1, wherein the nucleotide sequenceencoding the Cas9 nuclease variant is codon-optimized for the organismfrom which the cell to be modified is derived.
 12. The system of claim1, wherein the guide RNA is a single guide RNA (sgRNA).
 13. A method forgenetically modifying a cell, comprising: introducing the system ofclaim 1 to the cell, and thereby the Cas9 nuclease variant is targetedto the target sequence in the genome of the cell by the guide RNA, andresults in substitution, deletion and/or addition of one or morenucleotides in the target sequence.
 14. The method of claim 13, whereinthe cell is derived from mammals.
 15. The method of claim 13, whereinthe system is introduced into the cell by a method selected from:calcium phosphate transfection, protoplast fusion, electroporation,liposome transfection, microinjection, viral infection (such as abaculovirus, a vaccinia virus, an adenovirus and other viruses),particle bombardment, PEG-mediated protoplast transformation andagrobacterium-mediated transformation.
 16. The method of claim 14,wherein the mammal is a human, a mouse, a rat, a monkey, a dog, a pig, asheep, a cow or a cat, wherein the poultry is a chicken, a duck or agoose, and wherein the plant is rice, maize, wheat, sorghum, barley,soybean, peanut or Arabidopsis thaliana.